Cell products including metabolites and secreted enzymes such as exoenzymes are important biochemicals and enzymes that are produced by cells. Exoenzymes are any enzyme that is secreted by a cell for extracellular use. Metabolites are biochemicals produced by an organism that, unlike DNA or proteins, are not directly coded by the genome. Metabolites are common wastes like CO2, ethanol and urea but they may also be complex molecules with highly specialized functions such as quorum sensing, intercellular communication, microbial allelopathy and gene regulation. (i.e. Lactose and the lac operon).
Metabolites can be divided into primary and secondary metabolites. Primary metabolites include those biochemicals that are necessary for development, growth, and reproduction of the cell. These metabolites include amino acids and nucleotides. Secondary metabolites are those biochemicals that are not essential for cell metabolism. Secondary metabolites are low-molecular weight compounds that are synthesized by only some microbial strains and are often produced in connection with differentiation processes. In addition, they are often made as families of similar products. Some secondary metabolites include antibiotics, pigments, protease inhibitors, and toxins. Some examples of metabolites include citric acid and polysaccharides, which have application in the food industry as well as glutamic acid, which is a flavor enhancer.
The most famous microbial metabolite was discovered by a legendary accident. In 1928, Alexander Fleming plated Streptococcus bacteria on the surface of an agar plate and placed it by the window. By chance, a fungal spore landed on the plate and grew into a colony. Metabolites produced by the fungal colony diffused through the agar and killed the nearest Streptococcus bacteria. Fleming recognized this antibiotic action and won the Nobel Prize for discovering Penicillin.
In addition to penicillin, many other antibiotics are considered microbial metabolites including streptomycin and cephalosporin. In addition, immunosuppressants such as Cyclosporin A and cholesterol synthesis inhibitors such as Lovastatin are also considered microbial metabolites. Microbial exoenzymes such as glutaminase and asparginase have been shown to have anti-cancer activity.
A microbial colony consists of billions of identical, metabolically active cells. The growing colony is a metabolite factory and every well-isolated colony consists of a single species of microbe. Each species colony produces its own unique mixture of metabolites. After a colony is plated, a concentration gradient of soluble metabolites diffuses outward from the colony through the agar of the Petri plate.
Our invention comprises the use of metabolite transfer disks, which enable the cell-free spatially distinct and selective transfer of soluble biological molecules that are produced by microbial cell colonies. Metabolite transfer occurs via diffusion across a selectively permeable layer on the bottom of the magnetic metabolite transfer disk that is in contact with the cell colony. The specific composition of the gel layer in conjunction with the specific type of filter used for the selectively permeable bottom layer determines what cell products (or metabolites) are taken up into the gel layer or deposited on the filter. The metabolite transfer disks of the present invention allow scientists to safely isolate and transfer microbial metabolites and other biochemicals from cell colonies grown on the surface of Petri plates.
In another embodiment of the present invention, we provide for a diagnostic transfer disk that can be used to selectively isolate cell products while obtaining a metabolic profile using different indicator reagents. For over a century different species of microbes have been identified based on their distinct metabolic profiles. The metabolic profile is dependent on specific enzymes, which can be identified by specific indicators such as enzyme substrates that care catabolized to different colored products if active enzyme is present.
An example is the Caseinase Test, which identifies cell colonies that release the exoenzyme caseinase. These caseinase-positive colonies are identified by a clear zone that forms around them when grown on opaque skim milk agar. As the secreted caseinase diffuses through the milk agar, it breaks down the white milk protein casein into colorless peptides and amino acids that can then be easily absorbed by the cells. Accordingly, the present invention can facilitate cell free transfer and identification of biomolecules like exoenzymes while also supporting cell metabolic activity from above. Inclusion of extra nutrients or inducers, such as IPTG, into the transfer disk may be used to trigger a desired activity by the colony.
Other metabolic enzymes commonly assayed for include catalase, cytochrome oxidase, beta-glucuronidase (MUG), ONPG, nitrate reduction, starch hydrolysis, etc. Metabolic enzymes are usually revealed via a color reaction. Many reactions collectively sketch a biochemical profile that identifies species and strain. Some cellular enzymes are only released when cells lyse and die.
Identification of microbial species based on composite results of a battery of biochemical tests has been used for decades. Test kits like API 20E are routinely used to identify bacteria. Cells from a colony are usually inoculated into such tests with a dropper, loop or inoculation needle. The new invention conducts multiple biochemical assays on diffusible biomolecules transferred from cell colonies into the disk. Different indicator reagents can be used on each disk due to subdividing the gel layer into separate compartments. The compartmentalization of the present invention allows many different biomolecules to be diagnosed depending on the reagent used. This embodiment of the present invention can have expanded use as a wound dressing.
U.S. Pat. No. 6,333,093 to Burrell describes a multi-layered wound dressing with a semi transparent backing. Burrell's wound dressing changes color when an alcohol or electrolyte solution is added. This color change indicates successful activation of anti-microbial compounds present in the dressing itself. While a color change can be viewed through the bandage, Burrell's patent does not relate to the diagnostic identification of microbes or metabolites. It is not a diagnostic tool, but rather it is a direct applicator of chemotherapeutics. Other such bandages have been described which, like Burrell's invention, deliver drugs or therapeutics to the wound. Importantly, however, these do not selectively recover and identify specific biochemicals from the wound for the purpose of preliminary diagnosis and indication of infection as does the present invention.
The use of “smart bandages” gained popularity between the years of 2000 to 2005. These “smart bandages” normally have an electrical component used for diagnostic purposes. For instance, Miller and Fauchet developed a sand-grain sized wafer that differentiates between Gram-positive and Gram-negative bacteria thus showing that it was possible to accurately identify bacteria with a silicon sensor.
U.S. Pat. No. 7,226,733 to Chen et. al. disclosed a biological sensor having a porous semiconductor structure comprising a central layer interposed between upper and lower layers with each of the upper and lower layers having strata of alternating porosity. The biological sensor also has one or more probes coupled to the porous semiconductor structure. A detectable change occurs in a refractive index of the biological sensor upon binding of the one or more probes to the target molecule that can then be measured through photoluminescent emissions.
One embodiment of the present invention serves a similar function as the “smart bandages”; however, the present invention need not employ advanced electronics or sensors. The lack of advanced electronics or sensors is advantageous, particularly in regions where power is not available, such as in disaster areas. Also, rather than relying on probes for binding to specific targets such as membrane-integral lipids like Lipid-A which is present in the cell membrane of Gram negative bacteria, the present invention identifies infection and causative microbes based on enzymatic conversion of exogenous substrates in the disk into colored products. Other conversion techniques such as immunochromatographic antibody binding and metabolite dependent pH changes can be incorporated into the biochemical/microbial characterization wound dressing as well as the diagnostic transfer disks of the present invention. Unlike electronic bandages, these relatively simple techniques are generally known to those of ordinary skill in the art of microbiology.
The inventive device significantly advances the overall utility and demand for metabolite disks as well as provides new specialty transfer disks reported here for the first time. Cell free transfer of microbial cell colony metabolites via magnetic transfer disks is new and unobvious as is in-disk metabolic characterization and species identification and diagnosis. These features greatly expand the utility of the invention.